ABSTRACT: Background: Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most devastating pathogen of soybean. Many gene expression profiling studies have been conducted to investigate the responses of soybean to the infection by this pathogen using primarily the first-generation soybean genome array that covered approximately 37,500 soybean transcripts. However, no study has been reported yet using the second-generation Affymetrix soybean whole-genome transcript array (Soybean WT array) that represents approximately 66,000 predicted soybean transcripts. Results: In the present work, the gene expression profiles of two soybean plant introductions (PIs) PI 437654 and PI 567516C (both resistant to multiple SCN races) and cultivar (cv.) Magellan (susceptible to SCN) were compared in the presence or absence of the SCN inoculum at different time points (3 and 8 days post-inoculation). For this purpose, the Soybean WT array, which covers many more predicted soybean transcripts than the first-generation Affymetrix soybean array, was employed. Data analysis revealed that the two resistant soybean lines showed distinctive gene expression profiles from each other and from Magellan not only in response to the SCN inoculation, but also in the absence of SCN. Overall, 1,413 genes and many pathways, such as defense and hormonal pathways, were revealed to be differentially regulated. Among them, 297 genes were constitutively regulated in the two resistant lines (compared with cv. Magellan) and 1,146 genes were responsive to the SCN inoculation in the three lines, with 30 genes regulated both constitutively and by SCN. In addition to the findings similar to those in the published work, many genes involved in ethylene, protein degradation, and phenylpropanoid pathways were also revealed differentially regulated in the present study. GC-rich elements (e.g., GCATGC) were found over-represented in the promoter regions of certain groups of genes. These have not been observed before, and could be new defense-responsive regulatory elements. Conclusions: Different soybean lines showed different gene expression profiles in the presence and absence of the SCN inoculum. Both inducible and constitutive gene expression may contribute to resistance to multiple SCN HG Types in the resistant soybean PI lines. Ethylene, protein degradation, and phenylpropanoid pathways, as well as many other pathways reported previously, may play important roles in mediating the soybean-SCN interactions. The revealed genes, pathways, and promoter elements can be further explored to regulate or engineer soybean for resistance to SCN. Three soybean lines were compared in the study: Magellan (susceptible to SCN), PI 437654 (PI654; resistant to SCN), and PI 567516C (PI516C; resistant to SCN). Overall, thirty samples, including two biological replicates for each sample, were analyzed.